Abstract Viral skin infections are a significant cause of morbidity and mortality in the human populations, with millions of individuals infected worldwide with the poxvirus molluscum contagiosum virus (MCV), which is only held in check by the host immune system as immunocompromised individuals present with debilitating systemic infection. The related poxvirus Vaccinia virus (VACV) was used to immunize hundreds of millions of people during the smallpox eradication program, and remains a backbone of the most widely used viral vaccine vectors. During virus infection there is a significant change in the morphology of a target organ caused by cellular fusion during progression of the virus life cycle, as well as by the influx of a large variety of responding immune cells. The inflammation produced by infection can also stimulate the differentiation of non-classical populations of resident or infiltrating cells that do not resemble cells typically present in the steady state. Therefore, the complex phenotyping of these cells requires the use of large panels of probes (mostly antibodies). However, traditional methods (microscopy, flow cytometry, proteomics or genomics) do not allow simultaneous analysis of protein expression by individual cells, quantification and topological localization of cells and proteins relative to each other without damaging and confounding tissue dissociation, or analysis of 50-100 parameters from a single tissue. In order to accomplish this investigation we will perform non-invasive analysis of 50+ parameters using a Toponome Imaging System, an imaging cycler microscope that is a fully automated microscope that allows the spatial resolution of large molecular and cellular systems displaying many thousands of potential interactions in intact tissues. We will analyze the molecular and cellular changes in the skin at different points after infection of the skin of mice with using VACV (a model for poxvirus infection). We hypothesize that multiplexed toponomic analysis will reveal a unique molecular and cellular signature poxviral skin infection. In Aim 1 we will optimize staining protocols and examine and validate infection, localization and phenotype of resident and infiltrating cell populations during the initiation, control and resolution phases of VACV dermal infection. In Aim 2 we will examine the role of two individual meyloid cell populations in establishing both the morphology of the VACV lesion and the function of infiltrating cells. We anticipate that the results gained from these studies will form the basis of a future RO1 or PO1 proposal in which the mechanisms by which infected, resident and infiltrating cell populations interact in viral skin infections, with potential for the use of clinical samples to validate out studies in humans.